本文選題：豬博卡病毒 + 重組桿狀病毒�。� 參考：《山東大學》2016年博士論文

【摘要】：豬博卡病毒(Porcine Bocavirus, PBoV),是細小病毒科(Family Parvoviridae)、細小病毒亞科(Subfamily Parvoviridae)、博卡病毒屬(Bocavirus Genus)成員之一,最早在2009年,由瑞典科學家,在患有離乳后、多系統(tǒng)衰竭綜合癥(Post-weaning multisystemic wasting syndrome, PMWS)的豬淋巴結中發(fā)現(xiàn),之后,世界各國也陸續(xù)發(fā)現(xiàn)了此種病毒。到現(xiàn)在為止,PBoV感染已在瑞典、中國、美國、加拿大、墨西哥、羅馬尼亞、匈牙利、烏干達、韓國、日本等國家均有報道。PBoV是無包膜、二十面體對稱、單鏈DNA病毒。病毒顆粒的直徑25-30nm�；蚪M全長約5000bp,包含3個主要開放讀碼框架(open reading frames, ORFs), ORF1、ORF2、ORF3。ORF1,位于基因組5’端,編碼非結構蛋白NS1。ORF1含有保守序列,與病毒的滾環(huán)復制、解旋酶、以及ATP酶活性有關。ORF2,位于基因組3’端,編碼兩個主要的衣殼蛋白VP1、VP2,參與病毒顆粒的組裝,決定抗原性。ORF3編碼功能未知的非結構蛋白NP1,研究報道此蛋白是犬微小病毒(Canine minute virus, CMV)復制的必需蛋白。也有報道HBoV的NP1基因,可以阻斷干擾素(IFN)的合成,可能參與免疫逃逸。PBoV的分類方法還沒有統(tǒng)一,按照VP1基因核苷酸序列的不同,PBoV可分為三個基因組(Groups): PBoV G1、PBoV G2,和PBoV G3。其中PBoV G3又可以分成5個亞基因組(Subgroups)。到目前為止,關于PBoV的致病性的分析研究數(shù)據(jù)有限,但PMWS患病豬的PBoV的血清陽性率明顯高于非PMWS豬群,說明PBoV可能與PMWS相關。另外,PBoV常與豬圓環(huán)病毒(Porcine circovirus, PCV)、豬細環(huán)病毒(Porcine torque teno virus, PTTV)、豬流行性腹瀉病毒(Porcine epidemic diarrhea virus, PEDV)等共感染,PBoV在淋巴結、血清、腸道等許多組織中被檢測到,說明病毒可能會引起其它方面的病變。目前,對單一PBoV的發(fā)病機理,還沒有明確的證據(jù)。在病毒檢測方面,目前已經(jīng)建立了幾種不同的PCR和定量PCR的方法,用于檢測病毒核酸。同時也可用間接免疫熒光檢測法、以及酶聯(lián)免疫法(Enzyme-linked Immuno Sorbent Assay, ELISA),來檢測病毒抗原。但由于缺乏特異性好、敏感性高的抗-PBoV抗體的檢測方法,大規(guī)模的PBoV流行病學調查尚未能很好的開展。因此,通過基因工程方法表達病毒抗原來檢測抗體,對研究病毒的抗原性和免疫原性、進而對于開展PBoV的流行病學調查、乃至病毒疫苗的研制都有重要意義。目前病毒蛋白可以通過細菌、酵母、昆蟲細胞、哺乳類動物細胞等多種表達系統(tǒng)來表達。細菌(主要是大腸桿菌)表達系統(tǒng),有操作簡單、產(chǎn)量高的優(yōu)點,但不能對蛋白進行糖基化修飾；酵母表達系統(tǒng),其產(chǎn)物與天然分子也還有一定差異；哺乳動物表達系統(tǒng),雖然產(chǎn)物接近天然蛋白,但產(chǎn)量很低。昆蟲細胞表達系統(tǒng),作為真核細胞(Eukaryocyte)表達系統(tǒng),表達產(chǎn)量非常高,產(chǎn)物的功能與結構與天然蛋白相近。在昆蟲細胞表達系統(tǒng)中,桿狀病毒(Baculovirus)表達系統(tǒng)已被廣泛應用于類病毒顆粒(Virus-like particles)的合成。其工作原理是：將外源目的基因,通過基因重組(Gene recombination)的方式,整合到多角體病毒(AcNPV)的多角體蛋白基因的啟動子下游,然后將重組的桿狀病毒(recombinant baculorirus)感染昆蟲細胞；當Recombinant baculovirus在昆蟲細胞內復制時,外源目的基因,可以利用多角體蛋白強悍的啟動子的啟動作用,在昆蟲細胞中得到大量表達。產(chǎn)生的目的蛋白,具備與天然蛋白質相類似的功能和結構,抗原性也非常接近。特別是在表達無包膜病毒的構造蛋白時,可以在昆蟲細胞內,自動裝配成類病毒顆粒(VLPs)。這種VLPs,不僅在結構上與原始病毒的結構相近,而且容易純化,可以獲得高產(chǎn)量、高純度的抗原,適用于血清抗體的檢測,以及病毒疫苗的開發(fā)研制。目的利用桿狀病毒表達系統(tǒng)(Baculovirus expressing system),在昆蟲Tn5細胞中表達PBoV的主要構造蛋白VP2,以期獲得PBoV的類病毒顆粒(PBoV-LPs)。以PBoV-LPs為抗原,建立酶聯(lián)免疫法(ELISA),檢測抗-PBoV抗體,進而探討PBoV的血清流行病學的特征。同時研究PBoV的免疫原性及免疫反應性,為PBoV疫苗的研制開發(fā)奠定基礎。也為未知病毒的抗原性的研究建立一套研究體系和研究模型。方法1.運用PCR法,擴增PBoV的VP2基因,將VP2基因克隆到TA克隆載體,并進行大量擴增。2.構建VP2基因轉移載體pVL1393-VP2,與線性化的桿狀病毒DNA,一起轉染昆蟲細胞Sf9,得到重組桿狀病毒Ac[VP2]。之后,在Sf9細胞中,使其大量擴增。3.將重組桿狀病毒Ac[VP2],感染BTL-Tn5B1-4 (Tn5)細胞,進行蛋白表達。動態(tài)觀察蛋白表達：將細胞內的蛋白、上清中的蛋白進行SDS-PAGE電泳,考馬氏亮藍(Coomassie blue)染色后,觀察細胞內蛋白的表達,以及培養(yǎng)上清中蛋白的分泌,以確定回收蛋白的時間。4.收集培養(yǎng)7天后細胞上清液,離心、4℃重懸過夜后,進行CsCl密度梯度離心純化蛋白。用蛋白測序方法,檢測蛋白質N端氨基酸序列,驗證VP2蛋白的準確性。Uranyl acetate染色后,電鏡下觀察類病毒顆粒。5.將PBoV-LPs免疫家兔,獲取抗PBoV-LPs抗體。利用該抗體和其它病毒顆粒的免疫反應,來確認PBoV-LPs與HBoVs、以及PCV2-LPs的交叉反應性。6.建立抗體檢測的方法,通過檢測家豬和野豬血清中抗PBoV IgG抗體,了解PBoV的流行病學狀況。7.檢測家豬和野豬血清PBoV的DNA,明確家豬和野豬中流行的PBoV的分子生物學特征。結果1.重組桿狀病毒AC[VP2]感染的Tn5細胞中,檢出PBoV VP2蛋白,其分子量約為61.5KDa,細胞內蛋白的表達高峰,在感染后第三天到第五天,之后開始遞減；上清液中的蛋白,在感染后第三天開始檢測到,之后逐漸增多；感染后第7天,上清液中蛋白量達到高峰。2.感染后第七天收集細胞培養(yǎng)上清液,CsCl密度梯度離心法純化蛋白。分子量約為61.5KDa的蛋白質集中在收集管8,9,10,密度為1.300g/cm3。電鏡下觀察到直徑約為30nm的類病毒顆粒,形狀和原始病毒相似。蛋白質序列分析顯示,類病毒顆粒的N端5個氨基酸序列,與PBoV VP2蛋白的序列完全一致。說明VLPs是由PBoV VP2基因表達而來,而且PBoV VP2蛋白可以自行裝配成類病毒顆粒(VLPs)。3.為驗證病毒顆粒內部是否包被了核酸,利用MagNA Pure LC Total NucleicAcid Isolation Kit (Roche Applied Science)試劑盒,從類病毒顆粒中提取核酸,然后將提取物進行瓊脂糖凝膠電泳,進行觀察。結果顯示顆粒內部未檢測到核酸。4.將病毒顆粒免疫家兔,進行ELISA試驗,檢測抗體的效價。比較免疫前后兔血清中的抗體,免疫前兔血清中未檢出抗體；免疫后的兔血清中,抗PBoV-LPsIgG抗體的滴度高達1：409,600,說明PBoV-LPs具有良好的免疫原性。5. PBoV與HBoVs的抗原性比較：通過ELISA法檢測抗PBoV-LPs抗體與HBoV-LPs、抗HBoV-LPs抗體與PBoV-LPs的交叉反應性,來比較PBoV與HBoVs的抗原性的差別。發(fā)現(xiàn)兔抗-PBoV IgG與HBoV1-LPs和HBoV2-LPs均反應,但不與HBoV3-LPs和HBoV4-LPs反應。而PBoV-LPs,可與兔抗-HBoV1,2,3,4-LPs IgG發(fā)生交叉反應,但弱于同源抗原抗體間的反應。說明PBoV可與HBoVs之間有相同的抗原決定簇存在。PBoV-LPs不與兔抗-PCV2-LPs IgG反應,PCV2-LPs也不與兔抗-PBoV-LPs IgG發(fā)生反應,說明PBoV與PCV2抗原性完全不同。6.家豬和野豬的抗PBoV IgG抗體攜帶率：以PBoV-LPs作為抗原進行ELISA試驗檢測抗體。檢測了194份家豬和259份野豬血清,其結果,家豬血清抗體陽性率高于90.7%(176/194),野豬血清抗體陽性率平均為59.5%(154/259),證明PBoV的感染在家豬和野豬中是很普遍的現(xiàn)象。7. PBoV的病毒核酸檢測：利用巢式PCR擴增PBoV NS1部分基因,調查家豬及野豬的病毒攜帶狀況。從259份野豬血清中共檢出7份血清PBoV DNA陽性,其中兵庫縣采集的血清中有4份,茨城縣2份、長崎縣1份。核苷酸序列分析結果表明,這7例病毒DNA屬于PBoV G3型基因組,并可以細分成4個亞型基因組,有明顯的地域區(qū)別。家豬血清樣本中,并未檢出病毒DNA,其原因在于,90%以上的家豬都已有抗體,病毒已被清除。結論1.本課題成功地利用桿狀病毒表達體系,合成了豬博卡病毒類病毒顆粒(PBoV-LPs),并完成類病毒顆粒的鑒定,包括蛋白的表達量、直徑、電鏡形態(tài)、分子量、顆粒密度、和免疫原性等生物學指標。PBoV-LPs豬博卡病毒類病毒顆粒具有天然VP2蛋白的特性及免疫原性,可作為抗原用于抗體的檢測以及疫苗的研制。2.應用純化的PBoV-LPs作抗原,建立了抗體檢測的ELISA方法。并用此方法對PBoV的抗原性進行了比較。發(fā)現(xiàn)PBoV可與HBoVs發(fā)生交叉反應,PBoV與PCV2的抗原性完全不同。3.對家豬和野豬的PBoV的感染狀況,進行了流行病學調查：發(fā)現(xiàn)在家豬和野豬中,PBoV的感染都很普遍。家豬的感染率更高,和飼養(yǎng)環(huán)境有密切關聯(lián)。從野豬標本中檢出的PBoV DNA序列分析表明,PBoV的感染存在多樣化、地域性差異等特點。
[Abstract]:The porcine Boka virus (Porcine Bocavirus, PBoV) is one of the members of the parvovirus (Family Parvoviridae), parvovirus subfamily (Subfamily Parvoviridae) and the genus Boka virus (Bocavirus Genus). It was first in 2009 and was a Swedish scientist with multiple system failure syndrome (Post-weaning multisystemic wasting). So far, PBoV infection has been reported in Sweden, China, the United States, the United States, Canada, Mexico, Romania, Hungary, Uganda, Korea, Japan and other countries, which have reported that.PBoV is a non membrane, twenty body symmetry, single strand DNA virus. The diameter 25-30nm. of the virus particles. The total length of the genome is about 5000bp, including 3 main open reading code frames (open reading frames, ORFs), ORF1, ORF2, ORF3.ORF1, located at the 5 'end of the genome, and the encoded non structural protein NS1.ORF1 contains a conservative sequence, which is related to the viral roll ring replication, the helicase, and ATP enzyme activity.ORF2, located at the 3' end of the genome, and encodes two main capsid eggs. White VP1, VP2, participate in the assembly of virus particles, determine the unstructured protein NP1 of unknown.ORF3 encoding function of antigenicity. It is reported that this protein is a necessary protein for the replication of the canine parvovirus (Canine minute virus, CMV). It also reports the NP1 gene of HBoV, which can block the synthesis of interferon (IFN) and may be involved in the classification of immune escape.PBoV. No unification, according to the nucleotide sequence of the VP1 gene, PBoV can be divided into three genomes (Groups): PBoV G1, PBoV G2, and PBoV G3. of which PBoV G3 can be divided into 5 subgenomes (Subgroups). S pigs, indicating that PBoV may be associated with PMWS. In addition, PBoV is often associated with porcine circovirus (Porcine circovirus, PCV), porcine circovirus (Porcine Torque teno virus, PTTV), porcine epidemic diarrhea virus, and so on in many tissues such as lymph nodes, serum, intestine and so on. At present, there is no clear evidence for the pathogenesis of a single PBoV. In the field of virus detection, several different methods of PCR and quantitative PCR have been established to detect virus nucleic acid. Meanwhile, indirect immunofluorescence and Enzyme-linked Immuno Sorbent As are also used. Say, ELISA), to detect virus antigens. But because of the lack of specific and sensitive detection methods of anti -PBoV antibody, large-scale PBoV epidemiological investigation has not been well carried out. Therefore, the antigenicity and immunogenicity of the virus are studied by gene engineering method to study the antigenicity and immunogenicity of the virus, and then to carry out PBoV Epidemiological investigation and even the development of viral vaccines are of great significance. At present, viral proteins can be expressed through a variety of expression systems such as bacteria, yeast, insect cells and mammalian cells. The bacteria (mainly Escherichia coli) express system, which has the advantages of simple operation and high yield, but can not be glycosylated; yeast The expression system of the mammalian expression system, although the product is close to the natural protein, is very low in production. The expression system of the insect cell expression system, as the eukaryotic cell (Eukaryocyte) expression system, is very high in expression, and the function and structure of the product are similar to that of natural protein. The Baculovirus expression system has been widely used in the synthesis of Virus-like particles. The principle of this system is to integrate the exogenous target gene into the promoter of the polyhedrosis (AcNPV) polyhedron gene (AcNPV) gene by gene recombination (Gene recombination) and then recombine the recombinant baculovirus. Virus (recombinant baculorirus) infects insect cells; when Recombinant baculovirus replicates in insect cells, the exogenous target gene can be expressed in insect cells by the activation of the strong promoter of polyhedron protein. The target protein produced in the insect cells has the function and structure similar to that of the natural protein. The antigenicity is also very close. Especially in the expression of the structural protein without the envelope virus, it can be automatically assembled into the virus like particles (VLPs) in the insect cells. This VLPs is not only structurally similar to the structure of the original virus, but also easy to purify, and can obtain high yield, high purity antigen, suitable for detection of serum antibody and disease. The development and development of the virus vaccine. Aim to express the major structural protein VP2 of PBoV in the insect Tn5 cells (Baculovirus expressing system) in order to obtain the PBoV virus like particles (PBoV-LPs). The enzyme linked immunoassay (ELISA) was established with PBoV-LPs as the antigen, and the anti -PBoV antibody was detected. Then the serological epidemic of PBoV was discussed. At the same time, study the immunogenicity and immunoreactivity of PBoV, lay the foundation for the development and development of PBoV vaccine, and set up a set of research system and research model for the study of the antigenicity of the unknown virus. Method 1. the VP2 gene of PBoV was amplified by PCR method, the VP2 gene was cloned into the TA cloning vector, and a large number of.2. were amplified to construct VP2 base. The transfer vector pVL1393-VP2, transfected with the linearized baculovirus DNA, transfected the insect cell Sf9 together and obtained the recombinant baculovirus Ac[VP2]., in Sf9 cells, the recombinant baculovirus Ac[VP2], infected with BTL-Tn5B1-4 (Tn5) cells, were amplified by a large number of.3., and the protein expression was observed dynamically: the protein in the cell, the supernatant in the supernatant, was observed dynamically. After SDS-PAGE electrophoresis, the protein was stained with martensim blue (Coomassie blue), the protein expression in the cells and the secretion of protein in the supernatant were observed to determine the time for the recovery of protein..4. was collected and cultured for 7 days after the culture of the supernatant. After the centrifugation, the protein was purified by CsCl density gradient centrifugation. The protein was purified by CsCl density gradient, and the protein sequencing method was used. The sequence of protein N terminal amino acid was detected, and the accuracy of VP2 protein was verified by.Uranyl acetate staining. Under electron microscope, the virus like particle.5. was observed to immunize the rabbit and obtain anti PBoV-LPs antibody. The antibody and other virus particles were used to confirm the PBoV-LPs and HBoVs, and the PCV2-LPs cross reactivity.6. to establish the antibody detection. Methods to detect the anti PBoV IgG antibody in the serum of domestic pigs and wild boar, to understand the epidemiological status of PBoV and to detect the DNA of PBoV in domestic pigs and wild boar serum, and to identify the molecular biological characteristics of PBoV in domestic pigs and wild boars. Results 1. the PBoV VP2 protein was detected in Tn5 cells infected by the recombinant baculovirus AC[VP2], and the molecular weight of the protein was about 61.5KDa. The expression peak of intracellular protein, after infection third days to fifth days after infection, began to decrease, the protein in the supernatant began to be detected at third days after infection and then gradually increased; seventh days after infection, the protein content in the supernatant reached the peak.2. infection after seventh days to collect cell culture supernatant and CsCl density gradient centrifugation method. Protein. The protein with a molecular weight of about 61.5KDa is concentrated in the collection tube 8,9,10. The density of the virus particles with a diameter of about 30nm under the 1.300g/cm3. electron microscope is similar to that of the original virus. The protein sequence analysis shows that the 5 amino acid sequence of the N terminal of the virus like particles is exactly the same as the sequence of PBoV VP2 protein. It shows that VLPs is PBoV VP. The 2 gene was expressed, and the PBoV VP2 protein could be assembled into a virus like particle (VLPs).3. to verify whether the virus particles were wrapped in the nucleic acid. The nucleic acid was extracted from the virus like particles by using the MagNA Pure LC Total NucleicAcid Isolation Kit (Roche) reagent box, and then the extract was carried out by agarose gel electrophoresis. The results showed that the virus particles were not detected by nucleic acid.4. in the particle, and the virus particles were immune to the rabbit. The ELISA test was carried out to detect the titer of the antibody. The antibody in the rabbit serum before and after immunization was compared. The antibody in the rabbit serum before immunization was not detected. The titer of anti PBoV-LPsIgG anti body was up to 1:409600 in the rabbit serum after the immunization, indicating that the PBoV-LPs has a PBoV-LPs. The antigenicity of good immunogenicity.5. PBoV and HBoVs: the cross reactivity of anti PBoV-LPs antibody and HBoV-LPs, anti HBoV-LPs antibody and PBoV-LPs by ELISA method, to compare the difference between PBoV and HBoVs. S can cross reaction with Rabbit anti -HBoV1,2,3,4-LPs IgG, but it is weaker than the reaction between homologous antigen antibody. It shows that PBoV can have the same antigenic determinant as HBoVs,.PBoV-LPs does not react with Rabbit anti -PCV2-LPs IgG, PCV2-LPs also does not react with Rabbit anti -PBoV-LPs IgG, saying that PBoV and antigenicity are completely different from swine and wild boar. The anti PBoV IgG antibody carrying rate: using PBoV-LPs as the antigen to test the antibody by ELISA test. 194 domestic pigs and 259 Bleno Choshi were detected. The results showed that the positive rate of serum antibody of domestic pigs was higher than 90.7% (176/194), and the positive rate of serum antibody of wild boar was 59.5% (154/259). It proved that the infection of PBoV was a common phenomenon in domestic pigs and wild boars. 7. PBoV virus nucleic acid detection: using the nested PCR to amplify the PBoV NS1 gene and investigate the virus carrying status of domestic pigs and wild boar. 7 serum PBoV DNA positive were detected from 259 wild boar serum, of which there were 4 serums in Hyogo County, 2 in Ibaraki county and 1 in Ibaraki county. The nucleotide sequence analysis showed that these 7 virus DNA belonged to the sera. The PBoV G3 genomes can be subdivided into 4 subtypes, and there are obvious regional differences. The virus DNA is not detected in the pig serum samples. The reason is that more than 90% of the domestic pigs have antibodies and the virus has been cleared. Conclusion the 1. subjects successfully used baculovirus system to synthesize the porcine Boka virus particles (PBoV-L Ps) and complete identification of virus like particles, including protein expression, diameter, electron microscope morphology, molecular weight, particle density, and immunogenicity, and other biological indicators.PBoV-LPs porcine Boka virus particles have the characteristics and immunogenicity of natural VP2 protein, which can be used as Antiogen for antibody detection and vaccine development and.2. application and purification. PBoV-LPs was used as an antigen and a ELISA method for antibody detection was established. The antigenicity of PBoV was compared by this method. It was found that PBoV could cross reaction with HBoVs. The antigenicity of PBoV and PCV2 was completely different from.3. to PBoV in domestic pigs and wild boars. Epidemiological investigation was carried out: the infection of PBoV was found in domestic pigs and wild boars. It is common that the infection rate of domestic pigs is higher and closely related to the feeding environment. The analysis of PBoV DNA sequence detected from the specimens of wild boar shows that the infection of PBoV has the characteristics of diversity, regional difference and so on.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.65;Q78

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